1. Field of the Invention
The present invention relates to artificial scaffolding material for retaining protein and use of the same, and more particularly, to artificial scaffolding material suitable for causing a plurality of species of proteins such as enzymes to function cooperatively or stepwise, and the use of the same.
2. Description of the Related Art
In general, to carry forward a series of reactions via cooperative or stepwise reactions of a plurality of enzymes, it is thought to be preferable that related enzymes are arranged in a given order.
For instance, to degrade and use a biological integrative structure such as cellulose, cooperative and stepwise reactions of a plurality of enzymes are necessary. A microorganism species that degrades cellulose is provided with a complex of multiple species of enzymes (cellulosome) for degrading cellulose, on the cell surface. A cellulosome is thought to effectively degrade crystalline cellulose that has a low-degradability, by being provided with a cellulose-binding protein that is bound by a plurality of cellulolytic enzymes.
Based on this knowledge, attempts have been made to artificially construct an enzymatic series with excellent reaction efficiency, by imitating the structure of a cellulosome and functionally arranging enzymes. For instance, a plurality of enzymes that use a cellulose-binding protein having a plurality of enzyme-binding domains derived from Clostridium josui or the like is disclosed (Japanese Patent Application Publication No. 2000-157282 and Japanese Patent Application Publication No. 2004-236504).
In addition, a technique in which mini-cellulosomes are secreted by Clostridium acetobutylicum is disclosed (S. Perret et al., J. Bacterol., 186 (1), 253-257 (2004)). Furthermore, there also is an attempt of connecting to the surface layer of yeast cell a protein that comprises a cohesin derived from Clostridium cellulovorans and a ZZ domain derived from Staphylococcus aureus of which are connected by a linker (Itoh et al., C106, Proceedings of the 71th Meeting of Society of Chemical Engineers, Japan (2006)).
However, according to the above prior art references, although enzymes can be arranged using a linear scaffolding protein, they do not necessarily allow these enzymes to be arranged at high density. In addition, since the technique of the above S. Perret et al. produces mini-cellulosomes that use a linear scaffolding protein by secretion outside the cell, it is difficult to have the enzymes exist with a high contact probability with respect to substrate. In addition, although the technique of the above Itoh et al. surface layer-displays a linear scaffolding protein using agglutinin, the high degree of accumulation of the scaffolding protein at the cell surface layer cannot be achieved.
Thus, all of the above prior techniques handled the individual linear scaffolding protein provided with a plurality of protein binding sites, in forms that are separately secreted or displayed on the cell surface layer. Moreover, the above patent references and Itoh et al. intended to control the placement focusing on the functional arrangement of a plurality of enzymes. With Itoh et al., although a plurality of species of enzymes can be bonded to a scaffolding protein, the placement of scaffolding proteins in the cell surface layer cannot be controlled.
Therefore, in the current situation, no artificial scaffold has been provided yet, in which a plurality of proteins such as enzymes are placed at a high density to effectively degrade a substrate by cooperative or stepwise action of these proteins. In addition, no artificial scaffolding material has been provided yet, in which linear scaffolding proteins are placed two-dimensionally. In addition, no protein complex material has been provided, which has been complexed using such artificial scaffolding materials.